Method for obtaining RNA aptamers based on shape selection

Abstract

RNA molecules are selected from a random sequence library for their ability to bind to a selecting nucleic acid structural element. Selection of RNA aptamers with extensive Watson-Crick complementarity to the nucleic acid ligand is precluded by inclusion of a blocking oligodeoxynucleotide in the binding phase of the selection protocol.

Claims

1. A method for obtaining an RNA aptamer that binds a structural element of a selecting nucleic acid molecule comprising:

(a) contacting an RNA population suspected of containing an RNA aptamer that binds the structural element with an amount of blocking oligodeoxynucleotides, each having a nucleotide sequence equivalent to the nucleotide sequence of the structural element, for a time and under buffer conditions sufficient to allow duplex formation between the blocking oligodeoxynucleotide and all members of the RNA population having a sequence complementary to that of the blocking oligodeoxynucleotide, to yield a candidate RNA population comprising free RNA molecules and blocked RNA-oligodeoxynucleotide duplexes;

(b) contacting the candidate RNA population with the selecting nucleic acid molecule for a time and under buffer conditions sufficient to allow the formation of a noncovalent complex comprising bound RNA and the selecting nucleic acid molecule;

(c) separating non-complexed RNA from the noncovalent complex;

(d) separating the bound RNA from the selecting nucleic acid molecule of the noncovalent complex to yield a selected RNA population; and

(e) repeating steps (b) through (d), wherein step (b) in the repetition is performed on the selected RNA population, for a number of cycles sufficient to yield a selected RNA population comprising a detectable amount of RNA aptamer.

2. The method of claim 1 wherein step (d) further comprises reverse transcribing the selected RNA population to yield a selected cDNA population; amplifying the selected cDNA population to yield an amplified selected cDNA population; and transcribing the amplified selected cDNA population to yield an amplified selected RNA population; and wherein step (b) in the repetition is performed on the amplified selected RNA population.

3. The method of claim 2 wherein the RNA population of step (a) is a synthetic RNA library.

4. The method of claim 3 wherein the synthetic RNA library comprises at least about 10.sup.6 different RNA molecules.

5. The method of claim 4 wherein the synthetic RNA library comprises at least about 10.sup.13 different RNA molecules.

6. The method of claim 2 wherein each RNA molecule of the synthetic RNA library comprises a different nucleotide sequence of between about 20-100 nucleotides.

7. The method of claim 6 wherein the unique nucleotide sequence is between about 40-70 nucleotides.

8. The method of claim 6 wherein the unique nucleotide sequence of each RNA molecule is flanked on each of its 5' and 3' ends by a commonly-shared promoter nucleotide sequence.

9. The method of claim 2 wherein the structural element comprises a stem-loop structure.

10. The method of claim 2 wherein the selecting nucleic acid molecule is an RNA molecule.

11. The method of claim 10 wherein the structural element comprises a structure selected from the group consisting a hair pin structure, of a stem-loop structure, a bulge-loop structure, a pseudoknot, a tetraplex structure, and a G-quartet.

12. The method of claim 2 wherein the noncovalent complex formed in step (b) results from binding interactions that substantially exclude duplex- or triplex-type base pairing interactions.

13. The method of claim 2 wherein the amount of blocking oligodeoxynucleotides comprises a plurality of overlapping oligodeoxynucleotides, each overlapping oligodeoxynucleotide comprising at least 20 nucleotides and having a nucleotide sequence equivalent to a portion of the nucleotide sequence of the structural element, such that each nucleotide position in the structural element corresponds to a nucleotide position in at least one of the overlapping oligodeoxynucleotides.

14. The method of claim 2 wherein step (b) is performed in the presence of about 1-100 mM Mg.sup.2+.

15. The method of claim 14 wherein step (b) is performed in the presence of about 20-50 mM Mg.sup.2+.

16. The method of claim 2 wherein the nucleotide sequence of a member of the amplified selected RNA population is determined prior to step (e).

17. The method of claim 1 wherein the selecting nucleic acid molecule is affixed to a column matrix prior to step (b).

18. The method of claim 17 wherein step (d) further comprises eluting the bound RNA from the column in the presence of a chelating agent.

19. The method of claim 18 wherein step (b) is performed in the presence of about 1-100 mM Mg.sup.2+, and wherein the chelating agent is about equimolar with the concentration of Mg.sup.2+.

20. The method of claim 17 wherein step (d) further comprises eluting the bound RNA from the column with an amount of competing nucleic acid effective to displace the bound RNA from the selecting nucleic acid molecule.

21. The method of claim 20 wherein the nucleotide sequence of the competing nucleic acid comprises the nucleotide sequence of the structural element of the selecting nucleic acid molecule.

22. The method of claim 17 further comprising a preselection procedure performed prior to step (a), comprising (i) contacting the RNA population with the column matrix prior to attachment of the selecting nucleic acid molecule to the column matrix, for a time and under buffer conditions sufficient to allow the formation of an RNA:column matrix complex; (ii) separating the non-complexed RNA population from the RNA:column matrix complex to yield a preselected RNA population; and (iii) eluting the complexed RNA from the column matrix; and wherein step (a) is performed on the preselected RNA population.

23. The method of claim 1 wherein the selecting nucleic acid molecule is detectably labelled, and wherein step (c) further comprises using non-denaturing gel electrophoresis to separate non-complexed RNA from the noncovalent complex comprising bound RNA and a detectably selecting nucleic acid molecule.

24. The method of claim 23 wherein step (d) further comprises electroeluting the noncovalent complex from the gel and dialyzing the electroeluted noncovalent complex.